Direct-to-library methods, systems, and compositions

This application is a continuation of U.S. patent application Ser. No. 17/323,843, filed May 18, 2021, which is a continuation of International Patent Application No. PCT/US2019/062488, filed Nov. 20, 2019, which claims the benefit of priority to U.S. Provisional Application No. 62/770,181, filed Nov. 21, 2018, all of which are hereby incorporated by reference in their entireties.

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Apr. 18, 2025, is named 47697-713_302_SL.xml and is 26,604 bytes in size.

Next generation sequencing (NGS) can be used to gather massive amounts of data about the nucleic acid content of a sample. It can be particularly useful for analyzing nucleic acids in complex samples, such as clinical samples. However, before using the NGS methods, a starting sample must often be extracted, which lowers nucleic acid recovery, delays sequencing, delays reporting of clinical calls, introduces errors, introduces bias, and often results in chemical waste requiring controlled handling. Errors and biases can affect results in many cases, such as when there are low abundance nucleic acids or target nucleic acids in patient samples. Furthermore, processes for generating nucleic acid libraries from DNA and RNA simultaneously have been difficult to develop.

There is a need for more efficient and accurate methods for detecting and quantifying nucleic acids as well as preparing nucleic acid libraries. This need can be seen, for example, with respect to low abundance nucleic acids or target nucleic acids in patient samples.

Current technologies for nucleic acid analysis often impede efficient sample processing. Many sample preparation methods introduce bias against certain GC-contents, fragment length, and/or structural form(s) of nucleic acids, which can decrease the efficiency of sample processing. Indeed, many have limited use in that they can only detect one nucleic acid form at a time. For example, with respect to DNA and RNA, most sample preparation methods require that a sample be divided so that DNA and RNA can be processed separately. Similarly, nucleic acids of different secondary or higher order structures require separate processing. In addition, sample preparation methods for nucleic acids (e.g., cell-free nucleic acids (cfNA)) require nucleic acids to be extracted before processing into a sequencing library because of concentration issues as well as inhibitory effects of the non-nucleic acid substances present in the biological samples containing these nucleic acids.

In the case of low quantity nucleic acids or low quality nucleic acids, treatment of nucleic acids before library generation can introduce yield losses and biases, and decreases the ability to recover nucleic acids for detection. Finally, samples containing low quantities of nucleic acids, or low quality nucleic acids, may not have sufficient material to permit detection of both RNA and DNA, or of any secondary or higher order structures, which could result in the possible loss of valuable information about the sample. Additionally processes such as improper handling of samples, nuclease treatment and other acts of human intervention may alter fragment length distribution profiles.

The present invention provides direct-to-library methods for generating a nucleic acid library that render a product incorporating such a nucleic acid library more specific and sensitive to low abundance, low quality nucleic acids, less sensitive to seasonal variation and sample shipping conditions. The present invention also provides greater ability to work with lower sample volumes, reduces sequence length bias, secondary structure bias, and GC biases, provides a reduced turnaround time, enables better quality control of materials, decreases the false positive rate of the target nucleic acid detection, and provides a lower cost of goods. The reduction in sequence length bias, secondary structure bias and GC bias allows insight into the actual distribution of these values in a sample. The present methods may eliminate the need for harmful and/or hazardous denaturing agents (e.g., guanidinium chloride, guanidinium thiocyanate). In addition, the present invention completely eliminates (e.g., phenol, chloroform, trizol, isopropanol) or lowers (e.g., ethanol) the quantities of harmful and/or hazardous chemicals required by all or some current processes of converting nucleic acids from the biological sample to a nucleic acid library. The present invention also allows better discrimination between the nucleic acid signal endogenous to a biological sample from that of a nucleic acid signal originating from environmental contamination during sample handling and processing.

A first aspect provides a method of generating a nucleic acid library from an initial sample, the method comprising, consisting of, or consisting essentially of:

The nucleic acid library is generated without extracting nucleic acids from the initial sample or spiked initial sample prior to generating the nucleic acid library from the initial sample. Thus, the method of generating the nucleic acid library from an initial sample comprises, consists of, or consists essentially of:

The nucleic acids from an initial sample can be single-stranded and/or double-stranded. In some embodiments, the nucleic acid library comprises, consists of, or consists essentially of a single-stranded nucleic acid library. In some embodiments, the nucleic acid library comprises, consists of, or consists essentially of a double-stranded nucleic acid library. In some embodiments, the nucleic acid library comprises, consists of, or consists essentially of a single-stranded nucleic acid library and double-stranded nucleic acid library.

In some embodiments, the one or more process control molecules comprises, consists of, or consists essentially of one or more of ID Spike(s), Spanks, and/or Sparks or GC Spike-in Panel (See, for example, U.S. Pat. No. 9,976,181, which is incorporated by reference in its entirety herein, including any drawings). In some embodiments, the one or more process control molecules comprises, consists of, or consists essentially of dephosphorylation control molecules, denaturation control molecules, nucleotide tailing control molecules, adapter attachment control molecules, degradation assessment molecules, and/or ligation control molecules. In some embodiments, the one or more process control molecules comprises, consists of, or consists essentially of one or more of ID Spike(s), Spanks, Sparks or GC Spike-in Panel, dephosphorylation control molecules, denaturation control molecules, and/or ligation control molecules.

In some embodiments, the one or more process control molecules comprises, consists of, or consists essentially of a plurality of synthetic nucleic acids. In some embodiments, the one or more process control molecules comprises, consists of, or consists essentially of synthetic nucleic acids with at least two different GC contents with a known input concentration. In some embodiments, the one or more process control molecules comprises, consists of, or consists essentially of synthetic nucleic acids with at least three different GC contents with a known input concentration.

In some embodiments, the at least two or at least three different GC contents each have GC-content between about 10% and about 50%. In some embodiments, the at least two or at least three different GC contents each have GC-content between about 5% and about 40%. In some embodiments, the at least two or at least three different GC contents each have GC-content between about 20% and about 80%, between about 30% and about 70%, between about 40% and about 60%, or between about 10% and about 90%.

In some embodiments, the at least three different GC contents comprise, consist of, or consist essentially of a first GC content that is between about 10% and about 40%, a second GC content that is between about 40% and about 60%, and a third GC content that is between about 60% and about 90%. In some embodiments, the at least three different GC contents comprise, consist of, or consist essentially of more than three GC contents, each GC content comprising, consisting of, or consisting essentially of a content that is between about 0% and 100%. Different GC contents may be uniformly distributed or non-uniformly distributed within respective ranges.

In some embodiments, generating the nucleic acid library from the initial sample further comprises, consists of, or consists essentially of:

In some embodiments, generating the nucleic acid library from the initial sample further comprises, consists of, or consists essentially of:

The order of some of the steps may be reversed. For example, in some embodiments, generating the nucleic acid library from the initial sample further comprises, consists of, or consists essentially of:

In some embodiments, generating the nucleic acid library from the initial sample further comprises, consists of, or consists essentially of:

In some embodiments, a 5′-end adapter sequence is attached by the use in step (e) of a polymerase that has non-templated activity and, subsequently in step (f), using a template switching reaction to attach a 5′-end adapter sequence.

As above, the order of the steps may be reversed so that generating the nucleic acid library from the initial sample further comprises, consists of, or consists essentially of:

In some embodiments, a 5′-end adapter sequence is attached by the use in step (e) of a polymerase that has non-templated activity and, subsequently in step (f), using a template switching reaction to attach a 5′-end adapter sequence.

In general, the steps set forth herein need not be in any particular order and some steps may be performed concurrently with others. For example, in some embodiments, attaching an adapter to one or both ends of the denatured nucleic acids to produce adapted nucleic acids can occur in the order of steps as set forth above. In some embodiments, attaching an adapter to one or both ends of the denatured nucleic acids to produce adapted nucleic acids can occur concurrently or concurrently with dephosphorylation when included in the method.

In some embodiments, separating the adapted nucleic acids comprises, consists of, or consists essentially of immobilizing the adapted nucleic acids. In some embodiments, immobilization occurs on magnetic beads. In some embodiments, immobilization occurs on a modified glass, beads with functionalized surface, modified capillary surfaces, and/or modified columns. In some embodiments, immobilization occurs on a column. In some embodiments, separating the adapted nucleic acids comprises, consists of, or consists essentially of purifying the adapted nucleic acids. In some embodiments, separating the adapted nucleic acids comprises, consists of, or consists essentially of precipitating the adapted nucleic acids.

In some embodiments, separating the adapted nucleic acids comprises, consists of, or consists essentially of using a 3′-end protected 3-end adapter. In some embodiments, separating the adapted nucleic acids comprises, consists of, or consists essentially of separating adapted nucleic acids from unadapted nucleic acids by digesting unadapted nucleic acids with a 3′end exonuclease, the adapted nucleic acids comprising, consisting of, or consisting essentially of a 3′-end protected 3-end adapter.

In some embodiments, attaching a 3′-end adapter comprises, consists of, or consists essentially of attaching with a splint oligonucleotide. In some embodiments, attaching a 3′-end adapter comprises, consists of, or consists essentially of ligating with a Splint-R ligase. In some embodiments, attaching a 3-end adapter comprises, consists of, or consists essentially of adding a ligase. In some embodiments adding RNase inhibitor improves stability of splint oligonucleotides or endogenous RNA. In some embodiments, an adapter is attached through a primer extension reaction performed with a polymerase comprising, consisting of, or consisting essentially of a DNA-dependent or RNA-dependent polymerase. In some embodiments an adapter is attached by a polymerase that has a non-templated activity.

In some embodiments, the splint oligonucleotide comprises, consists of, or consists essentially of a single-stranded oligonucleotide. In some embodiments, the splint oligonucleotide comprises single-stranded and double-stranded portions. In some embodiments, the splint oligonucleotide comprises, consists of, or consists essentially of a random hexamer or a random hexamer with a UMI. In some embodiments, the single-stranded oligonucleotide comprises, consists of, or consists essentially of a sequencing adapter or a part of sequencing adapter. In some embodiments, the single-stranded oligonucleotide comprises, consists of, or consists essentially of a random hexamer and a sequencing adapter or random hexamer and a part of sequencing adapter.

In some embodiments, the nucleic acids comprise, consist of, or consist essentially of DNA and/or RNA. In some embodiments, the nucleic acids comprise, consist of, or consist essentially of DNA. In some embodiments, the nucleic acids comprise, consist of, or consist essentially of RNA. In some embodiments, the nucleic acids comprise, consist of, or consist essentially of a hybrid RNA-DNA complex.

Some embodiments further comprise, consist of, or consist essentially of enriching nucleic acids. Some embodiments further comprise, consist of, or consist essentially of enriching nucleic acids for fragments of a certain length. In some embodiments, denaturation is used to further enrich nucleic acids or target nucleic acids. In some embodiments, denaturation comprises, consists of, or consists essentially of selective denaturation. In some embodiments, selective denaturation comprises, consists of, or consists essentially of one or more denaturation steps effective for the selection of fragments of a certain length and/or GC-content. In some embodiments, enriching for fragments of a certain length or length range may occur through the use of proteinases, detergents, heparin, hemolysis and plasma concentration.

In some embodiments, selective denaturation comprises, consists of, or consists essentially of incubation at selected or elevated temperatures. In some embodiments, the selective denaturation step comprises, consists of, or consists essentially of incubation at a temperature of about 45° C., at a temperature of about 50° C., at a temperature of about 55° C., at a temperature of about 60° C., at a temperature of about 65° C., at a temperature of about 70° C., at a temperature of about 75° C., at a temperature of about 80° C., at a temperature of about 85° C., at a temperature of about 90° C., at a temperature of about 95° C., at a temperature of about 100° C., at a temperature of about 105° C., at a temperature of about 110° C. In some embodiments, setting the temperature occurs at any of the denaturation steps such as, for example, without limitation, following dephosphorylation, preceding 3′-end ligation, before or after primer extension, and/or during an elution step.

In some embodiments, selective denaturation comprises, consists of, or consists essentially of incubation for a selected time. In some embodiments, the selected time comprises, consists of, or consists essentially of about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19, minutes about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, or about 60 minutes. In some embodiments, incubation occurs at any of the denaturation steps such as, for example, without limitation, following dephosphorylation, preceding 3′-end adapter attachment, primer extension and/or during an elution step.

In some embodiments, the initial sample comprises, consists of, or consists essentially of a reduced sample size. The initial sample size required for the method may be reduced in the respect that volume required for the reduced initial sample size may be reduced in comparison to the volume of a sample size required for a method that includes removal or extraction prior to generation of a nucleic acid library. In some embodiments, the initial sample size is below at least about 250 μL, or at least about 100 μL, about 90 μL, about 80 μL, about 70 μL, about 60 μL, about 50 μL, about 40 μL, about 30 μL, about 20 μL, about 15 μL, about 10 μL, about 5 μL, about 4 μL, about 3 μL, about 2 μL, about 1 μL, about 0.9 μL, about 0.8 μL, about 0.7 μL, about 0.6 μL, about 0.5 μL, about 0.4 μL, about 0.3 μL, about 0.2 μL, or about 0.1 μL.

A second aspect provides a method of determining abundance of nucleic acids in an initial sample comprising target nucleic acids, the method comprising, consisting of, or consisting essentially of:

The nucleic acid library is generated without extracting nucleic acids prior to generating the nucleic acid library from the initial sample. Thus, the method of generating the nucleic acid library from an initial sample comprises, consists of, or consists essentially of:

In some embodiments, generating the nucleic acid library from the spiked initial sample further comprises, consists of, or consists essentially of:

In some embodiments, generating the nucleic acid library from the spiked initial sample further comprises, consists of, or consists essentially of:

The order of some of the steps may be reversed. For example, in some embodiments, generating the nucleic acid library from the initial sample further comprises, consists of, or consists essentially of:

In some embodiments, generating the nucleic acid library from the spiked initial sample further comprises, consists of, or consists essentially of:

In some embodiments, a 5′-end adapter sequence is attached by the use in step (e) of a polymerase that has non-templated activity and, subsequently in step (f), using a template switching reaction to attach a 5′-end adapter sequence.

As above, the order of the steps may be reversed so that generating the nucleic acid library from the spiked initial sample further comprises, consists of, or consists essentially of:

In some embodiments, a 5′-end adapter sequence is attached by the use in step (e) of a polymerase that has non-templated activity and, subsequently in step (f), using a template switching reaction to attach a 5′-end adapter sequence.

In general, the steps set forth herein need not be in any particular order and some steps may be performed concurrently with others. For example, in some embodiments, attaching an adapter to one or both ends of the denatured nucleic acids to produce adapted nucleic acids can occur in the order of steps as set forth above. In some embodiments, attaching an adapter to one or both ends of the denatured nucleic acids to produce adapted nucleic acids can occur concurrently with dephosphorylation.

Some embodiments further comprise, consist of, or consist essentially of adding an anti-digoxigenin antibody. In some embodiments, the anti-digoxigenin antibody is added after the 3-end adapter is attached to the denatured or dephosphorylated nucleic acids and before an adapter is attached to the 3-end of the complementary strand or extension primer is hybridized. Some embodiments further comprise beads comprising, consisting of, or consisting essentially of an anti-digoxigenin antibody. Beads may be pulled down by, for example, pelleting on a magnet. In some embodiments, the anti-digoxigenin antibody is added during a separation step, annealing step, primary extension step, or second ligation step.

In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of host nucleic acids and/or non-host nucleic acids, e.g., microbial and/or pathogen nucleic acids, fetal or organ donor nucleic acids, host nucleic acids, and/or nucleic acids that may be administered into a host as a therapeutic or for any other reason. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of host nucleic acids. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least one microbe and/or pathogen. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least two different microbes and/or pathogens. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least five different microbes and/or pathogens. In some embodiments, non-host nucleic acids may have entered the host indirectly from the diet or in a drug; in such cases the non-host nucleic acid would not indicate the presence of a non-host organism harbored by a host. Without being limited by example, bovine DNA derived from food or porcine DNA from a medicine may be detected in a host.

In some embodiments, the microbes or pathogens comprise, consist of, or consist essentially of archaea, bacteria, yeast, fungi, molds, eukaryotes, viruses, protozoa and/or nematodes. In some embodiments, microbes comprise, consist of, or consist essentially of DNA viruses, RNA viruses, culturable bacteria, additional fastidious and unculturable bacteria, mycobacteria, and eukaryotic pathogens (See, Bennett J. E., D., R., Blaser, M. J. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases; Saunders, Philadelphia, PA, 2014; and Netter's Infectious Disease, 1st Edition, edited by Elaine C. Jong, MD and Dennis L. Stevens, MD, PhD (2015)), each of which is incorporated herein by reference in its entirety. In some embodiments, microbes comprise, consist of, or consist essentially of microbes set forth in https://www.ncbi.nlm.nih.gov/genome/microbes/or https://www.ncbi.nlm.nih.gov/biosample/, each of which is incorporated herein in its entirety.

In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 different microbes and/or pathogens. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least 20 different microbes and/or pathogens. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least 30 different microbes and/or pathogens. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least 40 different microbes and/or pathogens. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least 50 different microbes and/or pathogens. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least 60 different microbes and/or pathogens. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least 70 different microbes and/or pathogens. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least 80 different microbes and/or pathogens. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least 90 different microbes and/or pathogens. In some embodiments, the target nucleic acids comprise, consist of, or consist essentially of microbial and/or pathogen nucleic acids from at least 100 different microbes and/or pathogens.

In some embodiments, the method further comprises, consists of, or consists essentially of:

A recovery profile may encompass any losses or biases including degradation loss. It is recognized that particularly with RNA, degradation may result in losses and changes in profile due to degradation.

In some embodiments, abundance comprises, consists of, or consists essentially of a relative abundance. In some embodiments, abundance comprises, consists of, or consists essentially of absolute abundance. Abundance may also be corrected for the process bias using various control molecules to obtain biased-corrected abundance.

In some embodiments, the method further comprises, consists of, or consists essentially of normalizing abundance of microbial or pathogen nucleic acid by using a weighting factor. In some embodiments, the weighting factor is obtained by analyzing a raw measurement of a first plurality of synthetic nucleic acids and a raw measurement of a second plurality of synthetic nucleic acids in comparison with a known concentration of the first plurality of synthetic nucleic acids and a known concentration of the second plurality of synthetic nucleic acids.

In some embodiments, determining abundance comprises, consists of, or consists essentially of methods excluding extraction and/or removal for some microbes or pathogens and methods which include extraction and/or removal for other pathogens. Any combination may be used as known by one skilled in the art. In some embodiments, the method is drawn to one or more combinations set forth in the drawings provided herein.